Differences in intratumor innate lymphoid cell composition between orthotopic and spontaneous pancreatic mouse models.

Pancreatic cancer remains an unmet medical need. Late diagnosis and the lack of efficient treatment significantly impact the prognosis of patients suffering from pancreatic cancer. Improving patient outcomes requires a deeper comprehension of the tumor ecosystem. To achieve this, a thorough exploration of the tumor microenvironment using pre-clinical models that accurately replicate human disease is imperative, particularly in understanding the dynamics of immune cell subsets. Surprisingly, the impact of model variations on the composition of the tumor microenvironment has been largely neglected. In this study, we introduce an orthotopic model of pancreatic ductal adenocarcinoma and a spontaneous model of insulinoma. Our findings reveal striking differences in the innate lymphoid cell infiltrate, highlighting the importance of considering model-specific influences when investigating the tumor microenvironment.

Ex vivo activation of the GCN2 pathway metabolically reprograms T cells, leading to enhanced adoptive cell therapy.

The manipulation of T cell metabolism to enhance anti-tumor activity is an area of active investigation. Here, we report that activating the amino acid starvation response in effector CD8+ T cells ex vivo using the general control non-depressible 2 (GCN2) agonist halofuginone (halo) enhances oxidative metabolism and effector function. Mechanistically, we identified autophagy coupled with the CD98-mTOR axis as key downstream mediators of the phenotype induced by halo treatment. The adoptive transfer of halo-treated CD8+ T cells into tumor-bearing mice led to robust tumor control and curative responses. Halo-treated T cells synergized in vivo with a 4-1BB agonistic antibody to control tumor growth in a mouse model resistant to immunotherapy. Importantly, treatment of human CD8+ T cells with halo resulted in similar metabolic and functional reprogramming. These findings demonstrate that activating the amino acid starvation response with the GCN2 agonist halo can enhance T cell metabolism and anti-tumor activity.

Dabrafenib Alters MDSC Differentiation and Function by Activation of GCN2.

The effect of targeted therapeutics on anticancer immune responses is poorly understood. The BRAF inhibitor dabrafenib has been reported to activate the integrated stress response (ISR) kinase GCN2, and the therapeutic effect has been partially attributed to GCN2 activation. Because ISR signaling is a key component of myeloid-derived suppressor cell (MDSC) development and function, we measured the effect of dabrafenib on MDSC differentiation and suppressive activity. Our data showed that dabrafenib attenuated MDSC ability to suppress T-cell activity, which was associated with a GCN2-dependent block of the transition from monocytic progenitor to polymorphonuclear (PMN)-MDSCs and proliferative arrest resulting in PMN-MDSC loss. Transcriptional profiling revealed that dabrafenib-driven GCN2 activation altered metabolic features in MDSCs enhancing oxidative respiration, and attenuated transcriptional programs required for PMN development. Moreover, we observed a broad downregulation of transcriptional networks associated with PMN developmental pathways, and increased activity of transcriptional regulons driven by Atf5, Mafg, and Zbtb7a. This transcriptional program alteration underlies the basis for PMN-MDSC developmental arrest, skewing immature MDSC development toward monocytic lineage cells. In vivo, we observed a pronounced reduction in PMN-MDSCs in dabrafenib-treated tumor-bearing mice suggesting that dabrafenib impacts MDSC populations systemically and locally, in the tumor immune infiltrate. Thus, our data reveal transcriptional networks that govern MDSC developmental programs, and the impact of GCN2 stress signaling on the innate immune landscape in tumors, providing novel insight into potentially beneficial off-target effects of dabrafenib. SIGNIFICANCE: An important, but poorly understood, aspect of targeted therapeutics for cancer is the effect on antitumor immune responses. This article shows that off-target effects of dabrafenib activating the kinase GCN2 impact MDSC development and function reducing PMN-MDSCs in vitro and in vivo. This has important implications for our understanding of how this BRAF inhibitor impacts tumor growth and provides novel therapeutic target and combination possibilities.

The intestinal microbiota modulates the transcriptional landscape of iNKT cells at steady-state and following antigen exposure.

Invariant Natural Killer T (iNKT) cells are unconventional T cells that respond to microbe-derived glycolipid antigens. iNKT cells exert fast innate effector functions that regulate immune responses in a variety of contexts, including during infection, cancer, or inflammation. The roles these unconventional T cells play in intestinal inflammation remain poorly defined and vary based on the disease model and species. Our previous work suggested that the gut microbiota influenced iNKT cell functions during dextran sulfate sodium-induced colitis in mice. This study, shows that iNKT cell homeostasis and response following activation are altered in germ-free mice. Using prenatal fecal transplant in specific pathogen-free mice, we show that the transcriptional signatures of iNKT cells at steady state and following αGC-mediated activation in vivo are modulated by the microbiota. Our data suggest that iNKT cells sense the microbiota at homeostasis independently of their T cell receptors. Finally, iNKT cell transcriptional signatures are different in male and female mice. Collectively, our findings suggest that sex and the intestinal microbiota are important factors that regulate iNKT cell homeostasis and responses. A deeper understanding of microbiota-iNKT cell interactions and the impact of sex could improve the development of iNKT cell-based immunotherapies.

Dabrafenib alters MDSC differentiation and function by activation of GCN2.

The effect of targeted therapeutics on anti-cancer immune responses is poorly understood. The BRAF inhibitor dabrafenib has been reported to activate the integrated stress response (ISR) kinase GCN2, and the therapeutic effect has been partially attributed to GCN2 activation. Since ISR signaling is a key component of myeloid-derived suppressor cell (MDSC) development and function, we measured the effect of dabrafenib on MDSC differentiation and suppressive activity. Our data showed that dabrafenib attenuated MDSC ability to suppress T cell activity, which was associated with a GCN2-dependent block of the transition from monocytic progenitor to polymorphonuclear (PMN)-MDSCs and proliferative arrest resulting in PMN-MDSC loss. Transcriptional profiling revealed that dabrafenib-driven GCN2 activation altered metabolic features in MDSCs enhancing oxidative respiration, and attenuated transcriptional programs required for PMN development. Moreover, we observed a broad downregulation of transcriptional networks associated with PMN developmental pathways, and increased activity of transcriptional regulons driven by Atf5 , Mafg , and Zbtb7a . This transcriptional program alteration underlies the basis for PMN-MDSC developmental arrest, skewing immature MDSC development towards monocytic lineage cells. In vivo , we observed a pronounced reduction in PMN-MDSCs in dabrafenib-treated tumor-bearing mice suggesting that dabrafenib impacts MDSC populations systemically and locally, in the tumor immune infiltrate. Thus, our data reveals transcriptional networks that govern MDSC developmental programs, and the impact of GCN2 stress signaling on the innate immune landscape in tumors, providing novel insight into potentially beneficial off target effects of dabrafenib.

Molecular, metabolic, and functional CD4 T cell paralysis in the lymph node impedes tumor control.

CD4 T cells are central effectors of anti-cancer immunity and immunotherapy, yet the regulation of CD4 tumor-specific T (TTS) cells is unclear. We demonstrate that CD4 TTS cells are quickly primed and begin to divide following tumor initiation. However, unlike CD8 TTS cells or exhaustion programming, CD4 TTS cell proliferation is rapidly frozen in place by a functional interplay of regulatory T cells and CTLA4. Together these mechanisms paralyze CD4 TTS cell differentiation, redirecting metabolic circuits, and reducing their accumulation in the tumor. The paralyzed state is actively maintained throughout cancer progression and CD4 TTS cells rapidly resume proliferation and functional differentiation when the suppressive constraints are alleviated. Overcoming their paralysis established long-term tumor control, demonstrating the importance of rapidly crippling CD4 TTS cells for tumor progression and their potential restoration as therapeutic targets.

Molecular, metabolic and functional CD4 T cell paralysis impedes tumor control.

CD4 T cells are important effectors of anti-tumor immunity, yet the regulation of CD4 tumor-specific T (T TS ) cells during cancer development is still unclear. We demonstrate that CD4 T TS cells are initially primed in the tumor draining lymph node and begin to divide following tumor initiation. Distinct from CD8 T TS cells and previously defined exhaustion programs, CD4 T TS cell proliferation is rapidly frozen in place and differentiation stunted by a functional interplay of T regulatory cells and both intrinsic and extrinsic CTLA4 signaling. Together these mechanisms paralyze CD4 T TS cell differentiation, redirecting metabolic and cytokine production circuits, and reducing CD4 T TS cell accumulation in the tumor. Paralysis is actively maintained throughout cancer progression and CD4 T TS cells rapidly resume proliferation and functional differentiation when both suppressive reactions are alleviated. Strikingly, Treg depletion alone reciprocally induced CD4 T TS cells to themselves become tumor-specific Tregs, whereas CTLA4 blockade alone failed to promote T helper differentiation. Overcoming their paralysis established long-term tumor control, demonstrating a novel immune evasion mechanism that specifically cripples CD4 T TS cells to favor tumor progression.

The transcription factor IRF2 drives interferon-mediated CD8+ T cell exhaustion to restrict anti-tumor immunity.

Type I and II interferons (IFNs) stimulate pro-inflammatory programs that are critical for immune activation, but also induce immune-suppressive feedback circuits that impede control of cancer growth. Here, we sought to determine how these opposing programs are differentially induced. We demonstrated that the transcription factor interferon regulatory factor 2 (IRF2) was expressed by many immune cells in the tumor in response to sustained IFN signaling. CD8+ T cell-specific deletion of IRF2 prevented acquisition of the T cell exhaustion program within the tumor and instead enabled sustained effector functions that promoted long-term tumor control and increased responsiveness to immune checkpoint and adoptive cell therapies. The long-term tumor control by IRF2-deficient CD8+ T cells required continuous integration of both IFN-I and IFN-II signals. Thus, IRF2 is a foundational feedback molecule that redirects IFN signals to suppress T cell responses and represents a potential target to enhance cancer control.

Pre-encoded responsiveness to type I interferon in the peripheral immune system defines outcome of PD1 blockade therapy.

Type I interferons (IFN-Is) are central regulators of anti-tumor immunity and responses to immunotherapy, but they also drive the feedback inhibition underlying therapeutic resistance. In the present study, we developed a mass cytometry approach to quantify IFN-I-stimulated protein expression across immune cells and used multi-omics to uncover pre-therapy cellular states encoding responsiveness to inflammation. Analyzing peripheral blood cells from multiple cancer types revealed that differential responsiveness to IFN-Is before anti-programmed cell death protein 1 (PD1) treatment was highly predictive of long-term survival after therapy. Unexpectedly, IFN-I hyporesponsiveness efficiently predicted long-term survival, whereas high responsiveness to IFN-I was strongly associated with treatment failure and diminished survival time. Peripheral IFN-I responsive states were not associated with tumor inflammation, identifying a disconnect between systemic immune potential and 'cold' or 'hot' tumor states. Mechanistically, IFN-I responsiveness was epigenetically imprinted before therapy, poising cells for differential inflammatory responses and dysfunctional T cell effector programs. Thus, we identify physiological cell states with clinical importance that can predict success and long-term survival of PD1-blocking immunotherapy.

3D microgels to quantify tumor cell properties and therapy response dynamics.

Tumors contain heterogeneous and dynamic populations of cells that do not all display the fast-proliferating properties that traditional chemotherapies target. There is a need therefore, to develop novel treatment strategies that target diverse tumor cell properties. Identifying therapy combinations is challenging however. Current approaches have relied on cell lines cultured in monolayers with treatment response being assessed using endpoint metabolic assays, which although enable large-scale throughput, do not capture tumor heterogeneity. Here, a 3D in vitro tumor model using micro-molded hydrogels (microgels), the Gels for Live Analysis of Compartmentalized Environments (GLAnCE) platform, is adapted into a 96-well plate format (96-GLAnCE) that integrates patient-derived organoids (PDOs) and is combined with longitudinal automated imaging to address these limitations. Using 96-GLAnCE, two measures of tumor aggressiveness are quantified, tumor cell growth and in situ regrowth after drug treatment, in both cell lines and PDOs. The use of longitudinal image-based readouts enables the identification of tumor cell phenotypes with cell population and subpopulation resolution that cannot be detected by standard bulk-soluble assays. 96-GLAnCE is a versatile and robust platform that combines 3D-ECM based models, PDOs, and real-time assay readouts, to provide an additional tool for pre-clinical anti-cancer drug discovery for the identification of novel targets with translatable clinical significance.

Myeloid Responses to Extracellular Vesicles in Health and Disease.

Extracellular vesicles are mediators of cell-cell communication playing a key role in both steady-state and disease conditions. Extracellular vesicles carry diverse donor-derived cargos, including DNA, RNA, proteins, and lipids that induce a complex network of signals in recipient cells. Due to their ability to capture particulate matter and/or capacity to polarize and orchestrate tissue responses, myeloid immune cells (e.g., dendritic cells, macrophages, etc.) rapidly respond to extracellular vesicles, driving local and systemic effects. In cancer, myeloid-extracellular vesicle communication contributes to chronic inflammation, self-tolerance, and therapeutic resistance while in autoimmune disease, extracellular vesicles support inflammation and tissue destruction. Here, we review cellular mechanisms by which extracellular vesicles modulate myeloid immunity in cancer and autoimmune disease, highlighting some contradictory results and outstanding questions. We will also summarize how understanding of extracellular vesicle biology is being utilized for novel therapeutic and diagnostic applications.

Tryptophan-derived microbial metabolites activate the aryl hydrocarbon receptor in tumor-associated macrophages to suppress anti-tumor immunity.

The aryl hydrocarbon receptor (AhR) is a sensor of products of tryptophan metabolism and a potent modulator of immunity. Here, we examined the impact of AhR in tumor-associated macrophage (TAM) function in pancreatic ductal adenocarcinoma (PDAC). TAMs exhibited high AhR activity and Ahr-deficient macrophages developed an inflammatory phenotype. Deletion of Ahr in myeloid cells or pharmacologic inhibition of AhR reduced PDAC growth, improved efficacy of immune checkpoint blockade, and increased intra-tumoral frequencies of IFNγ+CD8+ T cells. Macrophage tryptophan metabolism was not required for this effect. Rather, macrophage AhR activity was dependent on Lactobacillus metabolization of dietary tryptophan to indoles. Removal of dietary tryptophan reduced TAM AhR activity and promoted intra-tumoral accumulation of TNFα+IFNγ+CD8+ T cells; provision of dietary indoles blocked this effect. In patients with PDAC, high AHR expression associated with rapid disease progression and mortality, as well as with an immune-suppressive TAM phenotype, suggesting conservation of this regulatory axis in human disease.

N6-Methyladenosine Reader YTHDF1 Promotes ARHGEF2 Translation and RhoA Signaling in Colorectal Cancer.

BACKGROUND & AIMS: N6-methyladenosine (m6A) governs the fate of RNAs through m6A readers. Colorectal cancer (CRC) exhibits aberrant m6A modifications and expression of m6A regulators. However, how m6A readers interpret oncogenic m6A methylome to promote malignant transformation remains to be illustrated. METHODS: YTH N6-methyladenosine RNA binding protein 1 (Ythdf1) knockout mouse was generated to determine the effect of Ythdf1 in CRC tumorigenesis in vivo. Multiomic analysis of RNA-sequencing, m6A methylated RNA immunoprecipitation sequencing, YTHDF1 RNA immunoprecipitation sequencing, and proteomics were performed to unravel targets of YTHDF1 in CRC. The therapeutic potential of targeting YTHDF1-m6A-Rho/Rac guanine nucleotide exchange factor 2 (ARHGEF2) was evaluated using small interfering RNA (siRNA) encapsulated by lipid nanoparticles (LNP). RESULTS: DNA copy number gain of YTHDF1 is a frequent event in CRC and contributes to its overexpression. High expression of YTHDF1 is significantly associated with metastatic gene signature in patient tumors. Ythdf1 knockout in mice dampened tumor growth in an inflammatory CRC model. YTHDF1 promotes cell growth in CRC cell lines and primary organoids and lung and liver metastasis in vivo. Integrative multiomics analysis identified RhoA activator ARHGEF2 as a key downstream target of YTHDF1. YTHDF1 binds to m6A sites of ARHGEF2 messenger RNA, resulting in enhanced translation of ARHGEF2. Ectopic expression of ARHGEF2 restored impaired RhoA signaling, cell growth, and metastatic ability both in vitro and in vivo caused by YTHDF1 loss, verifying that ARHGEF2 is a key target of YTHDF1. Finally, ARHGEF2 siRNA delivered by LNP significantly suppressed tumor growth and metastasis in vivo. CONCLUSIONS: We identify a novel oncogenic epitranscriptome axis of YTHDF1-m6A-ARHGEF2, which regulates CRC tumorigenesis and metastasis. siRNA-delivering LNP drug validated the therapeutic potential of targeting this axis in CRC.

The Akt-mTORC1 pathway mediates Axl receptor tyrosine kinase-induced mesangial cell proliferation.

Glomerulonephritis (GN), an important pathologic feature of many renal diseases, is frequently characterized by mesangial cell proliferation. We and others have previously shown that the TAM family receptor tyrosine kinases Axl, Mer, and Tyro-3 contribute to cell survival, proliferation, migration, and clearance of apoptotic cells (ACs); that Axl contributes to GN by promoting mesangial cell proliferation; and that small molecule inhibition of Axl ameliorates nephrotoxic serum-induced GN in mice. We now show that stimulation of renal mesangial cell Axl causes a modest increase in intracellular Ca2+ and activates NF-κB, mTOR, and the mTOR-containing mTORC1 complex, which phosphorylates the ribosomal protein S6. Axl-induction of Akt activation is upstream of NF-κB and mTOR activation, which are mutually codependent. Axl-induced NF-κB activation leads to Bcl-xl up-regulation. Axl is more important than Mer at mediating AC phagocytosis by mesangial cells, but less important than Mer at mediating phagocytosis of ACs by peritoneal macrophages. Taken together, our data suggest the possibility that Axl mediates mesangial cell phagocytosis of ACs and promotes mesangial cell proliferation by activating NF-κB and mTORC1.

Dynamic CD4+ T cell heterogeneity defines subset-specific suppression and PD-L1-blockade-driven functional restoration in chronic infection.

Inhibiting PD-1:PD-L1 signaling has transformed therapeutic immune restoration. CD4+ T cells sustain immunity in chronic infections and cancer, yet little is known about how PD-1 signaling modulates CD4+ helper T (TH) cell responses or the ability to restore CD4+ TH-mediated immunity by checkpoint blockade. We demonstrate that PD-1:PD-L1 specifically suppressed CD4+ TH1 cell amplification, prevents CD4+ TH1 cytokine production and abolishes CD4+ cytotoxic killing capacity during chronic infection in mice. Inhibiting PD-L1 rapidly restored these functions, while simultaneously amplifying and activating TH1-like T regulatory cells, demonstrating a system-wide CD4-TH1 recalibration. This effect coincided with decreased T cell antigen receptor signaling, and re-directed type I interferon (IFN) signaling networks towards dominant IFN-γ-mediated responses. Mechanistically, PD-L1 blockade specifically targeted defined populations with pre-established, but actively suppressed proliferative potential, with limited impact on minimally cycling TCF-1+ follicular helper T cells, despite high PD-1 expression. Thus, CD4+ T cells require unique differentiation and functional states to be targets of PD-L1-directed suppression and therapeutic restoration.

Inhibition of the BTK-IDO-mTOR axis promotes differentiation of monocyte-lineage dendritic cells and enhances anti-tumor T cell immunity.

Monocytic-lineage inflammatory Ly6c+CD103+ dendritic cells (DCs) promote antitumor immunity, but these DCs are infrequent in tumors, even upon chemotherapy. Here, we examined how targeting pathways that inhibit the differentiation of inflammatory myeloid cells affect antitumor immunity. Pharmacologic inhibition of Bruton's tyrosine kinase (BTK) and the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) or deletion of Btk or Ido1 allowed robust differentiation of inflammatory Ly6c+CD103+ DCs during chemotherapy, promoting antitumor T cell responses and inhibiting tumor growth. Immature Ly6c+c-kit+ precursor cells had epigenetic profiles similar to conventional DC precursors; deletion of Btk or Ido1 promoted differentiation of these cells. Mechanistically, a BTK-IDO axis inhibited a tryptophan-sensitive differentiation pathway driven by GATOR2 and mTORC1, and disruption of the GATOR2 in monocyte-lineage precursors prevented differentiation into inflammatory DCs in vivo. IDO-expressing DCs and monocytic cells were present across a range of human tumors. Thus, a BTK-IDO axis represses differentiation of inflammatory DCs during chemotherapy, with implications for targeted therapies.

Amino Acid Transport and Metabolism in Myeloid Function.

Regulation of amino acid availability and metabolism in immune cells is essential for immune system homeostasis and responses to exogenous and endogenous challenges including microbial infection, tumorigenesis and autoimmunity. In myeloid cells the consumption of amino acids such as arginine and tryptophan and availability of their metabolites are key drivers of cellular identity impacting development, functional polarization to an inflammatory or regulatory phenotype, and interaction with other immune cells. In this review, we discuss recent developments and emerging concepts in our understanding of the impact amino acid availability and consumption has on cellular phenotype focusing on two key myeloid cell populations, macrophages and myeloid derived suppressor cells (MDSCs). We also highlight the potential of myeloid-specific of amino acid transporters and catabolic enzymes as immunotherapy targets in a variety of conditions such as cancer and autoimmune disease discussing the opportunities and limitations in targeting these pathways for clinical therapy.

Pan-cancer analysis of longitudinal metastatic tumors reveals genomic alterations and immune landscape dynamics associated with pembrolizumab sensitivity.

Serial circulating tumor DNA (ctDNA) monitoring is emerging as a non-invasive strategy to predict and monitor immune checkpoint blockade (ICB) therapeutic efficacy across cancer types. Yet, limited data exist to show the relationship between ctDNA dynamics and tumor genome and immune microenvironment in patients receiving ICB. Here, we present an in-depth analysis of clinical, whole-exome, transcriptome, and ctDNA profiles of 73 patients with advanced solid tumors, across 30 cancer types, from a phase II basket clinical trial of pembrolizumab (NCT02644369) and report changes in genomic and immune landscapes (primary outcomes). Patients stratified by ctDNA and tumor burden dynamics correspond with survival and clinical benefit. High mutation burden, high expression of immune signatures, and mutations in BRCA2 are associated with pembrolizumab molecular sensitivity, while abundant copy-number alterations and B2M loss-of-heterozygosity corresponded with resistance. Upon treatment, induction of genes expressed by T cell, B cell, and myeloid cell populations are consistent with sensitivity and resistance. We identified the upregulated expression of PLA2G2D, an immune-regulating phospholipase, as a potential biomarker of adaptive resistance to ICB. Together, these findings provide insights into the diversity of immunogenomic mechanisms that underpin pembrolizumab outcomes.

Nuclear receptors, the aryl hydrocarbon receptor, and macrophage function.

Nuclear receptors (NRs) are key regulators of innate immune responses and tissue homeostasis. Evidence indicates that NRs significantly impact steady-state immune regulation, uptake and processing of apoptotic cells, tolerance induction, and control of inflammatory immunity. In this review, we describe our current understanding of the NR activity for balancing inflammation and tolerance, the signaling cascade inducing the NR activation and functional responses, and different mechanisms of the NR-driven immune effects in the context of autoimmune diseases. We further describe the ligand-activated transcription factor the aryl hydrocarbon receptor (AhR) that exhibits analogous functionality. Moreover, we will discuss the putative role of NRs and AhR in immune regulation and disease pathogenesis providing a rationale for therapeutic targeting as a unique opportunities in the clinical management of autoimmune diseases.